The dynamic behavior of chromatophores marks the transition from bands to spots in leopard geckos

Significance This study focuses on the establishment of the banded pattern of the leopard gecko hatchlings and the transition to spots in the adult. We demonstrate that iridophores are necessary for the formation of bands and that melanophores autonomously form spots in the absence of other chromatophores, both in the regenerating tail and in the Mack Super Snow color morph. We establish that this morph lacks xanthophores and iridophores due to a genetic mutation in the PAX7 transcription factor. With transcriptomic data, we confirm the expression of PAX7 in both xanthophores and iridophores as they differentiate. Our study provides insights into the regulatory mechanisms governing skin pigmentation in leopard geckos and enhances our understanding of color pattern formation in reptiles.

Sequences of the leopard gecko wild type PAX7 isoforms.The protein translation is provided.The starting site is highlighted in green, while the stop codon is in red.
Sequence of the leopard gecko Mack Super Snow PAX7 isoforms.The protein translation is provided.The starting site is highlighted in green, while the stop codon is in red.

Figure S1 .
Figure S1.Ontogenetic changes of the wild type leopard gecko coloration pattern during the first 35 weeks after hatching.

Figure S2 .
Figure S2.Transmission Electron Microscopy images of the leopard gecko skin.All images are from wild type animals unless otherwise specified.(A) Hatchling skin from the white head region.(B) Adult skin from the yellow region.(C) Adult skin from a dorsal black spot showing the presence of epidermal melanophores.The dotted line marks the epidermis-dermis boundary.(D) Dermal melanophore from the adult dorsal black spot.(E) Adult ventral skin where only collagen fibers are present.(F) Hatchling Mack Super Snow skin where only dermal and epidermal melanophores are present.iri: iridophores, cf: collagen fibers, n: nucleus, xa: xanthosomes with amorphous material possibly containing carotenoids, xl: xanthosomes with concentric lamellae possibly containing pteridines.Scale bars: 1 μm.

Figure S3 .
Figure S3.Fluorescent imaging of leopard gecko skin.(A) Transverse section of hatchling head white skin.Note in yellow the elongated shape of the iridophores within the dashed circle.(B) Transverse section of adult yellow skin.The section crosses the top of the scale.A magnification of the remaining iridophores is shown in the dotted inset.yellow: reflectance of the guanine crystals in the iridophores at 505 nm, green: collagen fibers, magenta: nuclei stained with TO-PRO-3.Scale bars: 100 μm (A), 200 μm (B).

Figure S4 .
Figure S4.RAMAN microscopy of the leopard gecko iridophores.The spectrum was measured on a cryosection of the hatchling head white skin at the location indicated in the inset.

Figure S5 .
Figure S5.Shed of an adult leopard gecko.The black spots are visible on the shed, which is of epidermal origin.

Figure S6 .
Figure S6.Semi-quantitative amplification of thyroid hormone receptors A and B. The dermis and the epidermis were disassociated following on overnight Dispase I incubation at 4 ºC.The DNA ladder ranges from 100 to 1000 bp (SmartLadder SF, Eurogentec).W: whole skin, D: dermis, E: epidermis, A: THRA, B: THRB, WT: wild type, MSS: Mack Super Snow.

Figure S7 .
Figure S7.Histology of the regenerated tail.(A) Adult leopard gecko with a regenerated tail.The dashed line marks the boundary of the regenerated part.(B) Magnification of the regenerated tail of the animal in (A).(C) Magnification of the regenerated tail of a juvenile gecko with a banded dorsal pattern.A patch of xanthophores is also visible.(D) Greater magnification of (B) where the presence of melanophores is obvious among the black spots.(E) Transverse section of a black spot from the regenerated tail skin imaged with transmitted and coaxial reflected illumination.(F) Transverse section of a white part of the regenerated tail skin imaged with transmitted and coaxial reflected illumination.Iridophores are absent in both sections.Scale bars: 1 cm (A), 0.5 cm (B,C), 200 μm (D), 20 μm (E,F).

Figure S8 .
Figure S8.Ontogenetic changes of the Mack Super Snow leopard gecko coloration pattern during the first 30 and 31 weeks after hatching.

Figure S9 .
Figure S9.Iridophores are present in the white bands of the Mack Snow (mss/+) hatchlings as evidenced in xylene-treated paraffin sections with transmitted (A) and coaxial reflected illumination (B) and transmission electron microscopy (C).In the grey magnification window, we can appreciate the absence of xanthophores near the epidermis.In the yellow magnification window, the iridophores are visible deep in the dermis.Scale bars: 20 μm (A, B), 5 μm (C).

Figure S10 .
Figure S10.Area of black spots in wild-type and Mack Super Snow leopard geckos.Green box plots correspond to wild type animals, and yellow ones to Mack Super Snow.The area was measured in mm 2 .

Figure S11 .
Figure S11.Co-segregation on Chromosome 2. Proportion (y-axis) of quality-filtered SNP/MNPs cosegregating with the Mack Super Snow locus on Chromosome 2. The proportion was calculated in overlapping windows of 1 Mb with a step of 100 Kb.

Figure S12 .
Figure S12.Thirteen nucleotide deletion in Chromosome 17 of Mack Super Snow.Alignment of the homozygous (mss/mss) and the heterozygous (mss/+) parental libraries to Chromosome 17.In light grey, the aligned reads and in dark grey, the coverage at this position of the Chromosome.

Figure S13 .
Figure S13.Leopard gecko PAX7 isoforms.(A) Position of the primers used for the amplification of the PAX7 isoforms in wild type and Mack Super Snow samples.(B) PAX7 isoforms sequenced after cloning.The number on the right indicates the number of clones.Only MSS6 was not found possibly due to its low abundance.(C) Detail of the 118-nucleotide insertion in MSS4/5/6, which corresponds to the beginning of intron 4. The length of the exons is not in scale.

Figure S14 .
Figure S14.Semi-quantitative amplification of PAX7 isoforms.The mRNA was extracted from embryos at the developmental stage 35.The DNA ladder ranges from 100 to 1000 bp (SmartLadder SF, Eurogentec).

Figure S15 .
Figure S15.Single-cell transcriptomic data from the wild type embryonic dorsal skin.(A) UMAP with cell type assignment.(B) Heatmap with the expression level of selected markers for each cell type.(C) UMAP representations displaying the expression of the selected markers in all cells.

Figure S16 .
Figure S16.WISH and UMAP for classical and novel chromatophore markers.Dorsal view of stage 35 wild type embryos used for WISH with probes for classical and novel markers of (A) xanthophores, (B) iridophores, and (C) melanophores, and the accompanying UMAP representations, showing the expression of these genes in the chromatophores of stage 40 wild type embryos.Note the faint expression detected with the CALM1 probe in a region that would correspond to a yellow band on the hatchling.This result should be taken with caution.With the CRIP1 probe, the skeletal muscles are probably stained.Indeed, the single-cell transcriptomic analyses showed that, besides melanophores, this gene is also expressed in endothelial, muscle and immune (lymphoid) cells.Scale bars: 100 μm.

Figure S17 .
Figure S17.Graphical representation of the color pattern transition in wild type and Mack Super Snow leopard geckos.The number of iridophores is greatly reduced in the wild type adult geckos.Both xanthophores and iridophores are absent in Mack Super Snow geckos.Inspired by (52).M: melanophores, X: xanthophores, I: iridophores, (I): reduced number of iridophores.
H S I D G I L G A Q R G S P L L L aagaattggaccccctccccagcagacctacttcctcctaaactccgagggcgtacttct K N W T P S P A D L L P P K L R G R T S gcttggggagtcgctggtagtttccttctatggtaaccgcctggacgaaggctctgacgt A W G V A G S F L L W -P P G R R L -R cgagtctgaaccggacctgcccctgaagcgcaagcagcgccgcagccggaccaccttcac R V -T G P A P E A Q A A P Q P D H L H tgcggaacagttggaggagctggagaaggcctttgagaggacccattacccggatatcta C G T V G G A G E G L -E D P L P G Y L cacccgagaagagcttgcccagcggaccaagctcactgaggcccgggtccaggtctggtt H P R R A C P A D Q A H -G P G P G L V cagcaaccgaagagcacggtggcgtaagcaagcaggagccaaccagctagcagcttttaa Q Q P K S T V A -A S R S Q P A S S Fccacctcttgccagggggcttcccacccactgggatgccagctttgccaccctatcagct P P L A R G L P T H W D A S F A T L S A tccagattcgacctacccaacagccgccatttcccaagacgggggcagcaccgtgcacag S R F D L P N S R H F P R R G Q H R A Q accccagcctctcccgccgtccaccatgcaccaggggggattggcagccgccgccgccga T P A S P A V H H A P G G I G S R R R R ctccagctcagcgtatggggcccggcacagcttttccagctactcggacaccttcatgaa L Q L S V W G P A Q L F Q L L G H L H E ccctgcagctcccacaaaccacatgaaccccgtcagcaacggcctctcccctcaggtgat P C S S H K P H E P R Q Q R P L P S G D gagcatcctcagcaaccccagtggagtcccaccgcagccccagggcgacttttccatctc E H P Q Q P Q W S P T A A P G R L F H L ccccctccacggcagcttggacactgccaattccatctcggccagctgcagccagaggag P P T S C E A T S D L

Table S1 . Genomic DNA libraries used for mapping the Mack Super Snow causative variant.
In parentheses, we provide the total number of individuals in each library (column 'Source') and the average coverage for a 2.2 Gb genome (column 'Filtered reads').PE: Paired-end reads.

Table S2 . List of genes in the Mack Super Snow interval on Chromosome 17 based on the family mapping.
We provide information on the presence of non synonymous modifications in the protein coding part of the genes detected in the Mack Super Snow libraries when compared to the wild type.

Table S3 .
Genotyping for the 13-nucleotide deletion in PAX7 of the Mack Super Snow family individuals used for the mapping.

Table S4 .
Genotyping for the 13-nucleotide deletion in PAX7 of 190 individuals in our colony originating from 24 unrelated lineages obtained to study different morphs and from different breeders.Individuals found homozygous for the deletion are marked' as '-/-', heterozygous as '+/-' and those lacking the deletion as '+/+'.

Table S5 .
Primers used in this study.